Shiryaev Sergey A, Savinov Alexei Y, Cieplak Piotr, Ratnikov Boris I, Motamedchaboki Khatereh, Smith Jeffrey W, Strongin Alex Y
Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, California, United States of America.
PLoS One. 2009;4(3):e4952. doi: 10.1371/journal.pone.0004952. Epub 2009 Mar 20.
Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.
METHODOLOGY/PRINCIPAL FINDINGS: To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context.
CONCLUSIONS/SIGNIFICANCE: In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.
基质金属蛋白酶（MMPs）在髓鞘碱性蛋白（MBP）的片段化和脱髓鞘过程中起重要作用，这会导致自身免疫性多发性硬化症（MS）和实验性自身免疫性脑脊髓炎（EAE）。经典的MBP亚型主要在中枢神经系统的少突胶质细胞中表达。单一MBP基因的剪接变体（Golli-MBP BG21和J37）在神经元以及免疫细胞中广泛表达。各个MMPs对MBP切割的相对贡献尚不清楚。
方法/主要发现：为了阐明哪种MMP在切割MBP中起主要作用，我们通过体外切割反应，随后对切割片段进行质谱分析，确定了MMP-2、MMP-8、MMP-9、MMP-10、MMP-12、MT1-MMP、MT2-MMP、MT3-MMP、MT4-MMP、MT5-MMP和MT6-MMP对MBP、BG21和J37亚型的切割效率。结果，我们确定了MMP切割位点和所得片段的序列。我们发现MBP、BG21和J37对多种MMP蛋白水解高度敏感。然而，MT6-MMP（最初称为白细胞溶素）在切割MBP亚型方面优于所有其他MMPs。使用混合淋巴细胞培养试验，我们证明MT6-MMP对MBP亚型的蛋白水解很容易以接近定量的产量产生免疫原性N端1-15 MBP肽。该肽选择性地刺激从患有EAE的小鼠中分离出的、对MHC H-2(U)背景下呈现的1-15 MBP片段具有特异性的PGPR7.5 T细胞克隆的增殖。
结论/意义：总之，我们的生化观察结果使我们推测，由弗林蛋白酶激活并与脂筏相关的MT6-MMP在MS病理学中起重要作用，并且MT6-MMP是MS中一种新的且有前景的药物靶点，尤其是与其他单个MMPs相比时。
